Alpha-amylase variant with altered properties

ABSTRACT

The present invention relates to variants of parent alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, specific activity, and altered pI, in particular higher pI.

FIELD OF THE INVENTION

[0001] The present invention relates to variants (mutants) of parentalpha-amylases, in particular of Bacillus origin, which variant hasalpha-amylase activity and exhibits an alteration in at least one of thefollowing properties relative to said parent alpha-amylase: substratespecificity, substrate binding, substrate cleavage pattern, thermalstability, pH/activity profile, pH/stability profile, stability towardsoxidation, specific activity, and pI, in particular higher pI.

BACKGROUND OF THE INVENTION

[0002] Alpha-Amylases (alpha-1,4glucan-4-glucanohydrolases, E.C.3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis ofstarch and other linear and branched 1,4-glucosidic oligo- andpolysaccharides.

[0003] The object of the invention is to provide an improvedalpha-amylase, in particular suitable for detergent use.

BRIEF DISCLOSURE OF THE INVENTION

[0004] The object of the present invention is to provide analpha-amylases which variants in comparison to the corresponding parentalpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylaseactivity and exhibits an alteration in at least one of the followingproperties relative to said parent alpha-amylase: substrate specificity,substrate binding, substrate cleavage pattern, thermal stability,pH/activity profile, pH/stability profile, stability towards oxidation,Ca²⁺ dependency, specific activity, and pI.

Nomenclature

[0005] In the present description and claims, the conventionalone-letter and three-letter codes for amino acid residues are used. Forease of reference, alpha-amylase variants of the invention are describedby use of the following nomenclature:

[0006] Original amino acid(s): position(s): substituted amino acid(s)

[0007] According to this nomenclature, for instance the substitution ofalanine for asparagine in position 30 is shown as:

[0008] Ala30Asn or A30N

[0009] a deletion of alanine in the same position is shown as:

[0010] Ala30* or A30*

[0011] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0012] Ala30AlaLys or A30AK

[0013] A deletion of a consecutive stretch of amino acid residues, suchas amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

[0014] Where a specific alpha-amylase contains a “deletion” incomparison with other alpha-amylases and an insertion is made in such aposition this is indicated as:

[0015] *36Asp or *36D

[0016] for insertion of an aspartic acid in position 36.

[0017] Multiple mutations are separated by plus signs, i.e.:

[0018] Ala30Asp+Glu34Ser or A30N+E34S

[0019] representing mutations in positions 30 and 34 substitutingalanine and glutamic acid for asparagine and serine, respectively.

[0020] When one or more alternative amino acid residues may be insertedin a given position it is indicated as

[0021] A30N,E or

[0022] A30N or A30E

[0023] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

[0024] R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0025] Further, “A30X” means any one of the following substitutions:

[0026] A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K,A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is an alignment of the amino acid sequences of five parentalpha-amylases. The numbers on the extreme left designate the respectiveamino acid sequences as follows:

[0028] 1: SEQ ID NO: 6 (Bacillus licheniformis alpha-amylase)

[0029] 2: SEQ ID NO: 8 (KSM-AP1378 alpha-amylase)

[0030] 3: SEQ ID NO: 2 (KSM-K36 alpha-amylase)

[0031] 4: SEQ ID NO: 4 (KSM-K38 alpha-amylase)

DETAILED DISCLOSURE OF THE INVENTION

[0032] The object of the present invention is to provide analpha-amylases, in particular of Bacillus origin, which variants hasalpha-amylase activity and exhibits an alteration in at least one of thefollowing properties relative to said parent alpha-amylase: substratespecificity, substrate binding, substrate cleavage pattern, thermalstability, pH/activity profile, pH/stability profile, stability towardsoxidation, specific activity, and altered pI, in particular higher pI.

Parent Alpha-Amylases

[0033] Contemplated alpha-amylases include the alpha-amylases shown inSEQ ID NO: 2 or SEQ ID NO: 4 of Bacillus origin and alpha-amylase havingat least 70% identity thereto. The SEQ ID NO: 1 shows the DNA sequenceencoding KSM-K36 and SEQ ID NO: 3 show the DNA sequence encodingKSM-K38. These two alpha-amylases are disclosed in EP 1,022,334 (herebyincorporated by reference).

[0034] The KSM-K38 alpha-amylase has about 67% identity with theKSM-AP1378 alpha-amylase disclosed in WO 97/00324); 64% identity withthe #707 alpha-amylase derived from Bacillus sp.#707 disclosed byTsukamoto et al., Biochemical and Riophysical Research Communications,151 (1988), pp. 25-31; and about 63% identity with the Baciliuslicheniformis alpha-amylase described in EP 0252666 (ATCC 27811).

[0035] Other alpha-amylases within the scope of the present inventioninclude alpha-amylases i) which displays at least 70%, such as at least75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least97%, at least 99% homology with at least one of said amino acidsequences shown in SEQ ID NOS: 2 or 4, and/or ii) is encoded by a DNAsequence which hybridizes to the DNA sequences encoding theabove-specified alpha-amylases which are apparent from SEQ ID NOS: 1 or3.

[0036] In connection with property i), the homology may be determined asthe degree of identity between the two sequences indicating a derivationof the first sequence from the second. The homology may suitably bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package (described above). Thus, Gap GCGv8may be used with the following default parameters: GAP creation penaltyof 5.0 and GAP extension penalty of 0.3, default scoring matrix. GAPuses the method of Needleman/Wunsch/Sellers to make alignments.

[0037] Alternatively, the software Clustal X obtainable from EMBL(ftp-embl-heidelberg.de) may be used for multiple alignments with a gapcreation penalty of 30, a gap extension penalty of 1 without gappenalty.

[0038] A structural alignment between the KSM-K36 or KSM-K38alpha-amylases (SEQ ID NO: 2 and 4) and other alpha-amylase may be usedto identify equivalent/corresponding positions in other alpha-amylases.One method of obtaining said structural alignment is to use the Pile Upprogramme from the GCG package using default values of gap penalties,i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1.Other structural alignment methods include the hydrophobic clusteranalysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) andreverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1pp. 142-149 (1998). An alignment of the KSM-K36, KSM-K38, KSM-AP1378 andthe Bacillus licheniformis alpha-amylase is shown in FIG. 1.

Hybridisation

[0039] The oligonucleotide probe used in the characterisation of theKSM-K36 or KSM-K38 alpha-amylases in accordance with property ii) abovemay suitably be prepared on the basis of the full or partial nucleotideor amino acid sequence of the alpha-amylase in question.

[0040] Suitable conditions for testing hybridisation involve pre-soakingin 5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation inthe same solution supplemented with 100 mM ATP for 18 hours at 40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular_Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

[0041] In the present context, “derived from” is intended not only toindicate an alpha-amylase produced or producible by a strain of theorganism in question, but also an alpha-amylase encoded by a DNAsequence isolated from such strain and produced in a host organismtransformed with said DNA sequence. Finally, the term is intended toindicate an alpha-amylase, which is encoded by a DNA sequence ofsynthetic and/or cDNA origin and which has the identifyingcharacteristics of the alpha-amylase in question. The term is alsointended to indicate that the parent alpha-amylase may be a variant of anaturally occurring alpha-amylase, i.e., a variant, which is the resultof a modification (insertion, substitution, deletion) of one or moreamino acid residues of the naturally occurring alpha-amylase.

Altered Properties

[0042] The following discusses the relationship between mutations, whichare present in variants of the invention, and desirable alterations inproperties (relative to those a parent KSM-K36 or KSM-K38alpha-amylases), which may result therefrom.

[0043] As mentioned above the invention relates to alpha-amylasevariants with altered properties.

[0044] In an aspect the invention relates to variant with alteredproperties as mentioned above.

[0045] In the first aspect a variant of a parent KSM-K36 or KSM-K38alpha-amylase, comprising an alteration at one or more positions (usingSEQ ID NO: 2 or 4 for the amino acid numbering) selected from the groupof:

[0046]2,9,14,15,16,26,27,48,49,51,52,53,54,58,73,88,94,96,103,104,107,108,111,114,128,130,133,138,140,142,144,148,149,156,161,165,166,168,171,173,174,178,179,180,181,183,184,187,188,190,194,197,198,199,200,201,202,203,204,205,207,209,210,211,212,214,221,222,224,228,230,233,234,237,239,241,242,252,253,254,255,260,264,265,267,275,276,277,280,281,286,290,293,301,305,314,315,318,329,333,340,341,356,375,376,377,380,383,384,386,389,399,403,404,405,406,427,441,444,453,454,472,479,480

[0047] wherein

[0048] (a) the alteration(s) are independently

[0049] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position,

[0050] (ii) a deletion of the amino acid which occupies the position, or

[0051] (iii) a substitution of the amino acid which occupies theposition with a different amino acid,

[0052] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentalpha-amylase having the amino add sequence of the KSM-K36alpha-amylases shown in SEQ ID NO: 2.

[0053] In the KSM-38 alpha-amylase the target positions are:

[0054]2,9,14,15,16,26,27,48,49,51,52,53,54,58,73,88,94,96,103,104,107,108,111,114,128,130,133,138,140,142,144,148,149,156,161,165,166,168,171,173,174,178,179,180,181,183,184,187,188,190,194,197,198,199,200,201,202,203,204,205,207,209,210,211,212,214,221,222,224,228,230,233,234,237,239,241,242,252,253,254,255,260,264,265,267,275,276,277,280,281,286,290,293,301,305,314,315,318,329,333,340,341,356,375,376,377,380,383,384,386,389,399,403,404,405,406,427,441,444,453,454,472,479,480.

[0055] In a preferred embodiment of the invention the variant compriseone or more of the following substitutions (using SEQ ID NO: 2 for thenumbering):

[0056] G2P,A; M91,L,F; H14Y; L15M,I,F,T; E16P; H26Y,Q,R,N; D27N,S,T;G48A,V,S,T; N49X; Q51X; A52X; D53E,Q,R; V54X; A58V,L,I,F; V73L,I,F;E84Q; G88X; D94X; N96Q; M103I,L,F; N104D; M/L107G,A,V,T,S; G108A;F111G,A,V,I,L,T; A114D,I,L,M,V,R; T125S; D128T,E; S130T,C; Y133F,H;W138F,Y; G140H,R,K,D,N; D142H,R,K,N; S144P; N148S; A149I; R156H,K,D,N;N161X; W165R; D166E; R168P; E171L,I,F; H173R,K,L; I173L; L174I,F;A178N,Q,R,K,H; N179G,A,T,S; T180N,Q,R,K,H; N181X; N183X; W184R,K;D187N,S,T; E188P,T,I,S; N190F; D194X; L197X; G198X; S199X; N200X;I201L,M,F,Y; D202X; F203L,I,F,M; S204X;H205X; E207Y,R; Q209V,L,I,F,M;E210X; E211Q; L212I,F; D214N,R,K,H; D221N; E222Q,T; D224N,Q; Y228F;L230I,F; I233A,V,L,F; K234N,Q; P237X; W239X; T241L,I,F,M; S242P,R;A252T; D253G,A,V,N; Q254K; D255N,Q,E,P; G260A; K264Q,S,T; D265N,Y;V267L,I,F,M; D275N,T; E276K; M277T,I,L,F; E280N,T,Q,S; M281H,I,L,F;V286X, preferably V286Y,L,I,F; Y290X; Y293H,F; S301 G,A,D,K,E,R;R305A,K,Q,E,H,D,N; E314K,Q,R,S,T,H,N; A315K,R,S; I318L,M,F; T329S;E333Q; A340R,K,N,D,Q,E; D341P,T,S,Q,N; G356Q,E,S,T,A; S375P; A376S;K377L,I,F,M; M380I,L,F; E383P,Q; L384I,F; D386N,Q,R,K,I,L; Q389K,R;Y399A,D,H; W403X; D404N; I405L,F; V406I,L,F,A,D; N427X; H441 K,N,D,Q,E;R442Q; Q444E,K,R; A445V; Q448A; H453R,K,Q,N; A454S,T,P; G472R,N479Q,K,R; Q480K,R.

[0057] In another preferred embodiment of the invention the variantcomprise one or more of the following substitutions (using SEQ ID NO: 4for the numbering):

[0058] G2P,A; M9I,L,F; H14Y; L15M,I,F,T; E16P; H26Y,Q,R,N; D27N,S,T;G48A,V,S,T; N49X; Q51X; A52X; D53E,Q,R; V54X; A58V,L,I,F; V73L,I,F;E84Q; G88X; D94X; N96Q; M103I,L,F; N104D; M/L107G,A,V,T,S,I,L,F; G108A;F111G,A,V,I,L,T; A114D,I,L,M,V,R; T125S; D128T,E; S130T,C; Y133F,H;W138F,Y; G140H,R,K,D,N; D142H,R,K,N; S144P; N148S; A149I; R156H,K,D,N;N161X; W165R; D166E; R168P; E171L,I,F; H173R,K,L; I173L; I174L,F;A178N,Q,R,K,H; N179G,A,T,S; T180N,Q,R,K,H; N181X; N183X; W184R,K;D187N,S,T; E188P,T,I,S; N190F;D194X; L197X; G198X; S199X; N200X;I201L,M,F,Y; D202X;. F203L,I,F,M; S204X; H205X; E207Y,R; Q209V,L,I,F,M;D210X; D210E; E211Q; L212I,F; D214N,R,K,H; D221N; E222Q,T; D224N,Q;Y228F; L230I,F; I233A,V,L,F; K234N,Q; P237X; W239X; T241L,I,F,M;S242P,R; A252T; D253G,A,V,N; Q254K; D255N,Q,E,P; G260A; K264Q,S,T;D265N,Y; V267L,I,F,M; D275N,T; E276K; M277T,I,L,F; E280N,T,Q,S;M281H,I,L,F; V286X, preferably V286Y,L,I,F; Y290X; Y293H,F;S301G,A,D,K,E,R; R305A,K,Q,E,H,D,N; E314K,Q,R,S,T,H,N; A315K,R,S;M318L,I,F; T329S; E333,Q; A340R,K,N,D,Q,E; D341P,T,S,Q,N; G356Q,E,S,T,A;S375P; A376S; K377L,I,F,M; M380I,L,F; E383P,Q; L384I,F; D386N,Q,R,K,I,L;Q389K,R; Y399A,D,H; W403X; D404N; V405L,F,I; V406I,L,F,A,D; N427X;N441K,D,Q,E,H; R442Q; Q444E,K,R; A445V; Q448A; N453R,K,Q,H; G454A,S,T,P;G472R, N479Q,K,R; Q480K,R.

[0059] Within the scope of the invention are variants of other parentalpha-amylases (as defined herein) with one or more correspondingmutations.

[0060] In an embodiment of the invention a variant of the invention maycomprise the following combination of substitutions:

[0061] N49I+L/M107A;

[0062] N49L+L/M107A;

[0063] G48A+N49I+L/M 107A;

[0064] G48A+N49L+L/M107A;

[0065] E188S,T,P+N190F+I201F+K264S;

[0066] G48A+N49I+L/M107A+E188S,T,P+N190F+I201 F+K264S;

[0067] N190F+I201F;

[0068] N190F+K264S;

[0069] I201F+K264S;

[0070] G140H+D142H+R156H,Y(+S144P);

[0071] G140K+D142D+R156H,Y(+S144P);

[0072] L197M+G198Y+S199A;

[0073] L15T+E188S+Q209V+A376S+G472R;

[0074]G48A+N49I+L/M107A+G140H+D142H+R156H,Y+E188P+N190F+I201F+K264S(+144P);N49T+L/M107A+G140H+D142H+R156H,Y+E188P+N190F+I201F+K264S(144P); “/”before number indicate that KSM-K36 and KSM-K38 has different amino acidon the actual position. For instance L/M107A means that in KSM-K36 themutation is L107A and in KSM-K38 the substitution is M107A.

Altered pI

[0075] Important positions and mutations with respect to achievingaltered pI, in particular a higher pI, in particular at high pH (ie., pH8-10.5) include any of the positions and mutations listed in the in“Altered Properties” section. It should be noted that when thealpha-amylase of the invention is for detergent use a high pI isdesirable—for instance a pI in the range from 7-10.

Stability

[0076] Important positions and mutations with respect to achievingaltered stability, in particular improved stability (i.e., higher orlower) at especially high pH (i.e., pH 8-10.5) include any of thepositions and mutations listed in the in “Altered Properties” section.

Oxidation Stability

[0077] Variants of the invention may have altered oxidation stability,in particular higher oxidation stability, in comparison to the parentalpha-amylase.

[0078] In an embodiment such an alpha-amylase variant has one or more ofMethionine amino acid residues exchanged with any amino acid residueexcept for Cys and Met. Thus, according to the invention the amino acidresidues to replace the Methionine amino acid residue are the following:Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser,Thr, Trp, Tyr, and Val.

[0079] A preferred embodiment of the alpha-amylase of the invention ischaracterized by the fact that one or more of the Methionine amino acidresidues is (are) exchanged with a Phe, Leu, Thr, Ala, Gly, Ser, Ile, orVal amino acid residue, preferably a Leu, Ile, Phe amino acid residue.In this embodiment a very satisfactory activity level and stability inthe presence of oxidizing agents is obtained. Specifically this meansthat one or more of the Methionines in the following position may bereplaced or deleted using any suitable technique known in the art,including especially site directed mutagenesis and gene shuffling.Target Methionine positions, using the SEQ ID NO: 2 numbering (KSM-K36),are one or more of M7, M8, M103, N277, M281, M304, M383, M428, M438.

[0080] Target Methionine positions, using the SEQ ID NO: 4 numbering(KSM-K38), are one or more of M7, M8, M103, M107, M197, M277, M281,M304, M318, M383, M428, M438.

[0081] In a preferred embodiment of the mutant alpha-amylase of theinvention is characterized by the fact that the Methionine amino acidresidue at position M107 and/or M277 and/or M281, and/or M318 and/orM383 and/or M428 is(are) exchanged with any of amino acid residue expectfor Cys and Met, preferably with a Phe, Leu, Thr, Ala, Gly, Ser, Ile, orAsp. Also other parent alpha-amylases, as defined above, may have one ormore Methionines substituted or deleted in particular in correspondingpositions.

Specific Activity

[0082] Important positions and mutations with respect to obtainingvariants exhibiting altered specific activity, in particular increasedor decreased specific activity, especially at temperatures from 10-60°C., preferably 20-50° C., especially 30-40° C., include any of the belowpositions and substututions. The amino acid residues of particularimportance are those involved in substrate binding. Primary targetpositions, using the SEQ ID NO: 2 numbering (KSM-K36), are one or moreof G48, N49, Q51, A52, D53, V54, L107, G108, W165, D166, L197, G198,S199, K234, K264.

[0083] Primary target positions, using the SEQ ID NO: 4 numbering(KSM-K38), are one or more of G48, N49, Q51, A52, D53, V54, M107, G108,W165, D166, L197, G198, S199, K234, K264.

[0084] Preferred specific mutations/substitutions are the ones listedabove in the section “Altered Properties” for the positions in question.

Altered pH Profile

[0085] Important positions and mutations with respect to obtainingvariants with altered pH profile, in particular improved activity atespecially low pH (i.e., pH 4-6) include mutations of amino residueslocated close to the active site residues, i.e., D229, E261, D328.Primary target positions, using the SEQ ID NO: 2 numbering (KSM-K36),are one or more of N104, E333

[0086] Primary target positions, using the SEQ ID NO: 4 numbering(KSM-K38), are one or more of N104, E333.

[0087] Preferred specific mutations/substitutions are the ones listedabove in the section “Altered properties” for the positions in question.

Altered Alpha-Amylase Activity

[0088] A variant of the invention may have altered alpha-amylaseactivity, in particular increased alpha-amylase activity, in comparisonto the parent alpha-amylase using the Phadebas® assay described below inthe “Materials & Methods” section.

[0089] In a preferred embodiment of the invention an alpha-amylasesubstituted in a position corresponding to position 286 using the SEQ IDNO: 2 for the numbering has increased alpha-amylase activity. Preferredsubstitutions are V286Y,L,I,F.

[0090] In Bacillus sp. (SEQ ID NO: 2) the following substitution areresult in increased activity: V286X (i.e.,V286A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y), preferred are V286Y,L,I,F.

[0091] In Bacillus sp. (SEQ ID NO: 4) the following substitution areresult in increased activity: A286X (i.e.,V286R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V), preferred are A286Y,L,I,F.

Other Mutations

[0092] Other preferred mutations to increase the stability of aparticular protein include substitutions to a similar amino acid havinga larger side chain, in order to fill out internal holds in the globularstructure. Examples of these include glycine to alanine, alanine tovaline, valine to isoleucine or leucine, alanine to serine, serine tothreonine, asparagine to glutamine, asparatate to glutamate, phenyl totyrosine or tryptophane, tyrosine to tryptophane, asparagine orasparatate to histidine, histidine to tyrosine and lysine to argininesubstitutions, but are not limited to these examples only. Preferredmutations are Q84E, N96D, N121D, N393H, N444H.

[0093] Examples of larger mutations in SEQ 2 include: A315S/V and V101I.Examples of larger mutations in SEQ 4 include: V101I, A132V, D210E,A315SN, V408I, S416T and A447V. Also other parent alpha-amylases, asdefined above, may have one or more amino acid substituted into a largeramino acid, in particular in corresponding positions.

[0094] Other preferred mutations include substitutions of glycineresidues to decrease the flexibility of the protein backbone. Examplesof glycine substitutions in SEQ 2 and 4 includes: 19, 36, 48, 55, 57,65, 71, 82, 88, 99, 108, 131, 140, 145, 162, 191, 198, 216, 227, 260,268, 299, 300, 310, 332, 356, 357, 364, 368, 397, 410, 415, 423, 431,433, 432, 441, 447, 454, 457, 464, 466, 468, 474, 475 and in particularG464A/S/N. Also other parent alpha-amylases, as defined above, may haveone or more glycines substituted or deleted in particular incorresponding positions.

[0095] Other preferred mutations include introduction of prolineresidues in positions where possible with respect to limitations in thedihedral angles of the protein backbone and in the secondary structure.Examples of substitutions into proline in SEQ 2 include: W13, E16, Q51,L61, A109, G131, W182, D187, I233, I307, S334, W338, D341, W342, A379,S417.

[0096] Examples of substitutions into proline in SEQ 4 include: W13,E16, Q51, L61, A109, G131, S144, W182, D187, I233, I307, S334, W338,D341, W342, A379, S417. Also other parent alpha-amylases, as definedabove, may be stabilised by introduction of a proline, in particular incorresponding positions.

[0097] Important positions and mutations with respect to obtainingvariants with improved stability at low pH are Aspargine substitutions.Preferred mutations include substitution or deletion of one or moreAspargine (Asn). Target Aspargines in SEQ ID NO: 2 (KSM-36) are N4, N17,N23, N34, N49, N68, N93, N96, N104, N121, N124, N147, N148, N161, N172,N179, N181, N183, N190, N192, N200, N278, N289, N291, N306, N326, N360,N371, N373, N393, N421, N430, N455, N463, N473, N482, which may besubstituted with any other amino acid, or deleted, in particular N190F.

[0098] Target Aspargines in SEQ ID NO: 4 (KSM-38) are N17, N23, N49,N68, N93, N96, N104, N121, N124, N147, N148, N161, N172, N179, N181,N183, N190, N192, N200, N250, N278, N289, N291, N306, N326, N360, N371,N373, N393, 421, N430, N444, N455, N456, N463, N473, N482, which may besubstituted with any other amino acid, or deleted, in particular N190F.

[0099] Also other parent alpha-amylases, as defined above, may have oneor more Aspargines substituted or deleted in particular in correspondingpositions.

Methods for Preparing Alpha-Amylase Variants of the Invention

[0100] Several methods for introducing mutations into genes are known inthe art. After a brief discussion of the cloning ofalpha-amylase-encoding DNA sequences, methods for generating mutationsat specific sites within the alpha-amylase-encoding sequence will bediscussed.

Cloning a DNA Sequence Encoding an Alpha-Amylase

[0101] The DNA sequence encoding a parent alpha-amylase may be isolatedfrom any cell or microorganism producing the alpha-amylase in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the alpha-amylase to be studied.Then, if the amino acid sequence of the alpha-amylase is known,homologous, labeled oligonucleotide probes may be synthesized and usedto identify alpha-amylase-encoding clones from a genomic libraryprepared from the organism in question. Alternatively, a labeledoligonucleotide probe containing sequences homologous to a knownalpha-amylase gene could be used as a probe to identifyalpha-amylase-encoding clones, using hybridization and washingconditions of lower stringency.

[0102] Yet another method for identifying alpha-amylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming alpha-amylase-negative bacteriawith the resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for alpha-amylase, therebyallowing clones expressing the alpha-amylase to be identified.

[0103] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., The EMBO J. 3, 1984, pp. 801-805. In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[0104] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al., Science 239, 1988, pp. 487-491.

Site-Directed Mutagenesis

[0105] Once an alpha-amylase-encoding DNA sequence has been isolated,and desirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequ nces flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al., (1984,Biotechnology 2:646-639). U.S. Pat. No. 4,760,025 discloses theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette. However, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

[0106] Another method for introducing mutations intoalpha-amylase-encoding DNA sequences is described in Nelson and Long,Analytical Biochemistry 180, 1989, pp. 147-151. It involves the 3-stepgeneration of a PCR fragment, containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Random Mutagenesis

[0107] Random mutagenesis is suitably performed either as localised orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

[0108] The random mutagenesis of a DNA sequence encoding a parentalpha-amylase may be conveniently performed by use of any method knownin the art.

[0109] In relation to the above, a further aspect of the presentinvenbon relates to a method for generating a variant of a parentalpha-amylase, e.g. wherein the variant exhibits altered or increasedthermal stability relative to the parent, the method comprising:

[0110] (a) subjecting a DNA sequence encoding the parent alpha-amylaseto random mutagenesis,

[0111] (b) expressing the mutated DNA sequence obtained in step (a) in ahost cell, and

[0112] (c) screening for host cells expressing an alpha-amylase variantwhich has an altered property (e.g., pH-stability) relative to theparent alpha-amylase.

[0113] Step (a) of the above method of the invention is preferablyperformed using doped primers.

[0114] For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one that induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

[0115] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues. When such agents areused, the mutagenesis is typically performed by incubating the DNAsequence encoding the parent enzyme to be mutagenized in the presence ofthe mutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

[0116] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions, which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding thealpha-amylase enzyme by any published technique, using e.g. PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

[0117] Preferably, the doping is carried out using “constant randomdoping”, in which the percentage of wild-type and mutation in eachposition is predefined. Furthermore, the doping may be directed toward apreference for the introduction of certain nucleotides, and thereby apreference for the introduction of one or more specific amino acidresidues. The doping may be made, e.g., so as to allow for theintroduction of 90% wild type and 10% mutations in each position. Anadditional consideration in the choice of a doping scheme is based ongenetic as well as protein-structural constraints. The doping scheme maybe made using the DOPE program (see “Material and Methods” section),which, inter alia, ensures that introduction of stop codons is avoided.

[0118] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent alpha-amylase is subjectedto PCR under conditions that increase the mis-incorporation ofnucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp.11-15).

[0119] A mutator strain of E. coil (Fowler et al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding thealpha-amylase by, e.g., transforming a plasmid containing the parentglycosylase into the mutator strain, growing the mutator strain with theplasmid and isolating the mutated plasmid from the mutator strain. Themutated plasmid may be subsequently transformed into the expressionorganism.

[0120] The DNA sequence to be mutagenized may be conveniently present ina genomic or cDNA library prepared from an organism expressing theparent alpha-amylase. Alternatively, the DNA sequence may be present ona suitable vector such as a plasmid or a bacteriophage, which as suchmay be incubated with or otherwise exposed to the mutagenising agent.The DNA to be mutagenized may also be present in a host cell either bybeing integrated in the genome of said cell or by being present on avector harboured in the cell. Finally, the DNA to be mutagenized may bein isolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0121] In some cases it may be convenient to amplify the mutated DNAsequence prior to performing the expression step b) or the screeningstep c). Such amplification may be performed in accordance with methodsknown in the art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

[0122] Subsequent to the incubation with or exposure to the mutagenisingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which was carried the DNA sequence encoding the parentenzyme during the mutagenesis treatment. Examples of suitable host cellsare the following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus, and gram-negative bacteria such as E. coli orPseudomonas.

[0123] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

[0124] The random mutagenesis may be advantageously localized to a partof the parent alpha-amylase in question. This may, e.g., be advantageouswhen certain regions of the enzyme have been identified to be ofparticular importance for a given property of the enzyme, and whenmodified are expected to result in a variant having improved properties.Such regions may normally be identified when the tertiary structure ofthe parent enzyme has been elucidated and related to the function of theenzyme.

[0125] The localized, or region-specific, random mutagenesis isconveniently performed by use of PCR generated mutagenesis techniques asdescribed above or any other suitable technique known in the art.Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by insertion into a suitable vector,and said part may be subsequently subjected to mutagenesis by use of anyof the mutagenesis methods discussed above.

Alternative Methods of Providing Alpha-Amylase Variants

[0126] Alternative methods for providing variants of the inventioninclude gene-shuffling method known in the art including the methods,e.g., described in WO 95/22625 (from Affymax Technologies N.V.) and WO96/00343 (from Novo Nordisk ANS).

Expression of Alpha-Amylase Variants Expresion Vectors

[0127] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

[0128] The recombinant expression vector carrying the DNA sequenceencoding an alpha-amylase variant of the invention may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

Promoters

[0129] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thtranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E.coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

Transcription Terminators

[0130] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding thealpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

Replication Sequences

[0131] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

Selectable Markers

[0132] The vector may also comprise a selectable marker, e.g., a genethe product of which complements a defect in the host cell, such as thedal genes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

Secretion Sequences

[0133] While intracellular expression may be advantageous in somerespects, e.g., when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus alpha-amylases mentioned herein comprise a preregionpermitting secretion of the expressed protease into the culture medium.If desirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

Host Cells

[0134] The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

[0135] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of analpha-amylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[0136] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

[0137] Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such asE.coli or pseudomonas. The transformation of the bacteria may, forinstance, be effected by protoplast transformation or by using competentcells in a manner known per se.

[0138] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

Method of Producing an Alpha-Amylase Variant of the Invention

[0139] In a yet further aspect, the present invention relates to amethod of producing an alpha-amylase variant of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

[0140] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the alpha-amylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

[0141] The alpha-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

Industrial Applications

[0142] Owing to their activity at alkaline pH values, the alpha-amylasevariants of the invention are well suited for use in a variety ofindustrial processes, in particular the enzyme finds potentialapplications as a component in detergents, e.g., laundry, dishwashingand hard surface cleaning detergent compositions, but it may also beuseful for desizing of textiles, fabrics and garments, beer making orbrewing, in pulp and paper production, and further in the production ofsweeteners and ethanol (see for instance U.S. Pat. No. 5,231,017—herebyincorporated by reference), such as fuel, drinking and industrialethanol, from starch or whole grains.

Starch Conversion

[0143] Conventional starch-conversion processes, such as liquefactionand saccharification processes are described, e.g., in U.S. Pat. No.3,912,590 and EP patent publications Nos. 252,730 and 63,909, herebyincorporated by reference.

[0144] A “traditional” starch conversion process degrading starch tolower molecular weight carbohydrate components such as sugars or fatreplacers includes a debranching step.

Starch to Sugar Conversion

[0145] In the case of converting starch into a sugar the starch isdepolymerized. A such depolymerization process consists of aPre-treatment step and two or three consecutive process steps, viz. aliquefaction process, a saccharification process and dependent on thedesired end product optionally an isomerization process.

Pre-Treatment of Native Starch

[0146] Native starch consists of microscopic granules, which areinsoluble in water at room temperature. When an aqueous starch slurry isheated, the granules swell and eventually burst, dispersing the starchmolecules into the solution. During this “gelatinization” process thereis a dramatic increase in viscosity. As the solids level is 30-40% in atypically industrial process, the starch has to be thinned or“liquefied” so that it can be handled. This reduction in viscosity istoday mostly obtained by enzymatic degradation.

Liquefaction

[0147] During the liquefaction step, the long chained starch is degradedinto branched and linear shorter units (maltodextrins) by analpha-amylase. The liquefaction process is carried out at 105-110° C.for 5 to 10 minutes followed by 1-2 hours at 95° C. The pH lies between5.5 and 6.2. In order to ensure optimal enzyme stability under theseconditions, 1 mM of calcium is added (40 ppm free calcium ions). Afterthis treatment the liquefied starch will have a “dextrose equivalent”(DE) of 10-15.

Saccharification

[0148] After the liquefaction process the maltodextrins are convertedinto dextrose by addition of a glucoamylase (e.g., AMG™) and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase (e.g., Promozyme™) (U.S. Pat. No. 4,560,651). Before thisstep the pH is reduced to a value below 4.5, maintaining the hightemperature (above 95° C.) to inactivate the liquefying alpha-amylase toreduce the formation of short oligosaccharide called “panose precursors”which cannot be hydrolyzed properly by the debranching enzyme.

[0149] The temperature is lowered to 60° C., and glucoamylase anddebranching enzyme are added. The saccharification process proceeds for24-72 hours.

[0150] Normally, wh n denaturing the α-amylase after the liquefactionstep about 0.2-0.5% of the saccharification product is the branchedtrisaccharide 6²-alpha-glucosyl maltose (panose) which cannot bedegraded by a pullulanase. If active amylase from the liquefaction stepis present during saccharification (i.e., no denaturing), this level canbe as high as 1-2%, which is highly undesirable as it lowers thesaccharification yield significantly.

Isomerization

[0151] When the desired final sugar product is, e.g., high fructosesyrup the dextrose syrup may be converted into fructose. After thesaccharification process the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immmobilized glucoseisomerase (such as Sweetzyme™ IT).

Ethanol Production

[0152] In general alcohol production (ethanol) from whole grain can beseparated into 4 main steps

[0153] Milling

[0154] Liquefaction

[0155] Saccharification

[0156] Fermentation

Milling

[0157] The grain is milled in order to open up the structure andallowing for further processing. Two processes are used wet or drymilling. In dry milling the whole kernel is milled and used in theremaining part of the process. Wet milling gives a very good separationof germ and meal (starch granules and protein) and is with a fewexceptions applied at locations where there is a parallel production ofsyrups.

Liquefaction

[0158] In the liquefaction process the starch granules are solubilizedby hydrolysis to maltodextrins mostly of a DP higher than 4. Thehydrolysis may be carried out by acid treatment or enzymatically byalpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

[0159] Enzymatic liquefaction is typically carried out as a three-stephot slurry process. The slurry is heated to between 60-95□C, preferably80-85□C, and the enzyme(s) is (are) added. Then the slurry is jet-cookedat between 95-140□C, preferably 105-125□C, cooled to 60-95□C and moreenzyme(s) is (are) added to obtain the final hydrolysis. Theliquefaction process is carried out at pH 4.5-6.5, typically at a pHbetween 5 and 6. Milled and liquefied grain is also known as mash.

Saccharification

[0160] To produce low molecular sugars DP₁₋₃ that can be metabolized byyeast, the maltodextrin from the liquefaction must be furtherhydrolyzed. The hydrolysis is typically done enzymatically byglucoamylases, alternatively alpha-glucosidases or acid alpha-amylasescan be used. A full saccharification step may last up to 72 hours,however, it is common only to do a pre-saccharification of typically40-90 minutes and then complete saccharification during fermentation(SSF). Saccharification is typically carried out at temperatures from30-65□C, typically around 60□C, and at pH 4.5.

Fermentation

[0161] Yeast typically from Saccharomyces spp. is added to the mash andthe fermentation is ongoing for 24-96 hours, such as typically 35-60hours. The temperature is between 26-34□C, typically at about 32□C, andthe pH is from pH 3-6, preferably around pH 45.

[0162] Note that the most widely used process is a simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzyme isadded together. When doing SSF it is common to introduce apre-saccharification step at a temperature above 50□C, just prior to thefermentation.

Distillation

[0163] Following the fermentation the mash is distilled to extract theethanol.

[0164] The ethanol obtained according to the process of the inventionmay be used as, e.g., fuel ethanol; drinking ethanol, i.e., potableneutral spirits; or industrial ethanol.

By-Products

[0165] Left over from the fermentation is the grain, which is typicallyused for animal feed either in liquid form or dried.

[0166] Further details on how to carry out liquefaction,saccharification, fermentation, distillation, and recovering of ethanolare well known to the skilled person.

[0167] According to the process of the invention the saccharificationand fermentation may be carried out simultaneously or separately.

Pulp and Paper Production

[0168] The alkaline alpha-amylase of the invention may also be used inthe production of lignocellulosic materials, such as pulp, paper andcardboard, from starch reinforced waste paper and cardboard, especiallywhere re-pulping occurs at pH above 7 and where amylases facilitate thedisintegration of the waste material through degradation of thereinforcing starch. The alpha-amylase of the invention is especiallyuseful in a process for producing a papermaking pulp from starch-coatedprinted-paper. The process may be performed as described in WO 95/14807,comprising the following steps:

[0169] a) disintegrating the paper to produce a pulp,

[0170] b) treating with a starch-degrading enzyme before, during orafter step a), and

[0171] c) separating ink particles from the pulp after steps a) and b).

[0172] The alpha-amylases of the invention may also be very useful inmodifying starch where enzymatically modified starch is used inpapermaking together with alkaline fillers such as calcium carbonate,kaolin and clays. With the alkaline alpha-amylases of the invention itbecomes possible to modify the starch in the presence of the filler thusallowing for a simpler integrated process.

Desizing of Textiles, Fabrics and Garments

[0173] An alpha-amylase of the invention may also be very useful intextile, fabric or garment desizing. In the textile processing industry,alpha-amylases are traditionally used as auxiliaries in the desizingprocess to facilitate the removal of starch-containing size, which hasserved as a protective coating on weft yarns during weaving. Completeremoval of the size coating after weaving is important to ensure optimumresults in the subsequent processes, in which the fabric is scoured,bleached and dyed. Enzymatic starch breakdown is preferred because itdoes not involve any harmful effect on the fiber material. In order toreduce processing cost and increase mill throughput, the desizingprocessing is sometimes combined with the scouring and bleaching steps.In such cases, non-enzymatic auxiliaries such as alkali or oxidationagents are typically used to break down the starch, because traditionalalpha-amylases are not very compatible with high pH levels and bleachingagents. The non-enzymatic breakdown of the starch size does lead to somefiber damage because of the rather aggressive chemicals used.Accordingly, it would be desirable to use the alpha-amylases of theinvention as they have an improved performance in alkaline solutions.The alpha-amylases may be used alone or in combination with a cellulasewhen desizing cellulose-containing fabric or textile. Desizing andbleaching processes are well known in the art. For instance, suchprocesses are described in WO 95/21247, U.S. Pat. No. 4,643,736, EP119,920 hereby in corporate by reference.

[0174] Commercially available products for desizing include Aquazyme®and Aquazyme® Ultra from Novo Nordisk A/S.

Beer Making

[0175] The alpha-amylases of the invention may also be very useful in abeer-making process; the alpha-amylases will typically be added duringthe mashing process.

Detergent Compositions

[0176] The alpha-amylase of the invention may be added to and thusbecome a component of a detergent composition.

[0177] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

[0178] In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, a pectate lyase, and/or aperoxidase.

[0179] In general the properties of the chosen enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and nonenzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

[0180] Proteases: Suitable proteases include those of animal, vegetableor microbial origin. Microbial origin is preferred. Chemically modifiedor protein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtlisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin168 (described in WO 89/06279). Examples of trypsin-like pro-teases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89106270 and WO 94/25583.

[0181] Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235 and 274.

[0182] Preferred commercially available protease enzymes includeAlcalase®, Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (NovoNordisk A/S), Maxatase®, Maxacal, Maxapem®, Properase®, Purafect®,Purafect Oxp®, FN2®, and FN3® (Genencor International Inc.).

[0183] Lipases: Suitable lipases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Examples of useful lipases include lipases from Humicola (synonymThermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described inEP 258 068 and EP 305 216 or from H. insolens as described in WO96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P.pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase,e.g., from B. subtilis (Dartois et al. (1993), Biochemica et BiophysicaActa, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus(WO 91/16422).

[0184] Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

[0185] Preferred commercially available lipase enzymes include Lipolasemand Lipolase Ultra™ (Novo Nordisk A/S).

[0186] Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839. Examples of usefulalpha-amylases are the variants described in WO 94/02597, WO 94/18314,WO 96/23873, and WO 97/43424, especially the variants with substitutionsin one or more of the following positions: 15, 23, 105, 106, 124, 128,133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305,391, 408, and 444.

[0187] Commercially available amylases are Duramyl™, Termamyl™,Natalase™, Fungamyl™ and BAN™ (Novo Nordisk A/S), Rapidase™ andPurastar™ (from Genencor International Inc.).

[0188] Cellulases: Suitable cellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable cellulases include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.,the fungal cellulases produced from Humicola insolens, Myceliophthorathermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307,U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No.5,776,757 and WO 89/09259.

[0189] Especially suitable cellulases are the alkaline or neutralcellulases having colour care benefits. Examples of such cellu-lases arecellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO96/29397, WO 98/08940. Other examples are cellulase variants such asthose described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046,U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO98/12307 and PCT/DK98/00299.

[0190] Commercially available cellulases include Celluzyme™, andCarezyme™ (Novo Nordisk A/S), Clazinase®, and Puradax HA® (GenencorInternational Inc.), and KAC-500(B)® (Kao Corporation).

[0191] Peroxidases/Oxidases: Suitable peroxidases/oxidases include thoseof plant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

[0192] Commercially available peroxidases include Guardzyme® (NovoNordisk A/S).

[0193] Pectate lyase: Many pectate lyases have been described in theart, see e.g. WO 99/27083 (Novozymes A/S) or WO 99/27084 (NovozymesA/S), both of which are incorporated herein by reference in theirtotality.

[0194] The detergent enzyme(s) may be included in a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising all of these enzymes. Adetergent additive of the invention, i.e., a separate additive or acombined additive, can be formulated, e.g., granulate, a liquid, aslurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

[0195] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty adds. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric add according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

[0196] The detergent composition of the invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

[0197] The detergent composition comprises one or more surfactants,which may be non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic. The surfactants are typically present at alevel of from 0.1% to 60% by weight.

[0198] When included therein the detergent will usually contain fromabout 1% to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

[0199] When included therein the detergent will usually contain fromabout 0.2% to about 40% of a non-ionic surfactant such as alcoholethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside,alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide,fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, orN-acyl N-alkyl derivatives of glucosamine (“glucamides”).

[0200] The detergent may contain 0-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, tripho-sphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

[0201] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

[0202] The detergent may contain a bleaching system, which may comprisea H₂O₂ source such as perborate or percarbonate which may be combinedwith a peracid-forming bleach activator such astetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate.Alternatively, the bleaching system may comprise peroxyacids of, e.g.,the amide, imide, or sulfone type.

[0203] The enzyme(s) of the detergent composition of the inven-tion maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the com-position may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

[0204] The detergent may also contain other conventional detergentingredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil re-deposition agents, dy s, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

[0205] It is at present contemplated that in the detergent compositionsany enzyme, in particular the enzyme of the invention, may be added inan amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liquor, preferably 0.05-5 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

[0206] The enzyme of the invention may additionally be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference. Dishwash Deterget Compositions

[0207] The enzyme of the invention mat also be used in dish washdetergent compositions, including the following: 1) POWDER AUTOMATICDISHWASHING COMPOSITION Nonionic surfactant 0.4-2.5% Sodium metasilicate 0-20% Sodium disilicate  3-20% Sodium triphosphate 20-40% Sodiumcarbonate  0-20% Sodium perborate 2-9% Tetraacetyl ethylene diamine(TAED) 1-4% Sodium sulphate  5-33% Enzymes 0.0001-0.1%  

[0208] 2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-2%  (e.g. alcohol ethoxylate) Sodium disilicate 2-30% Sodium carbonate10-50%  Sodium phosphonate 0-5%  Trisodium citrate dihydrate 9-30%Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10%Tetraacetyl ethylene diamine (TAED) 1-2%  Polyacrylate polymer 6-25%(e.g. maleic acid/acrylic acid copolymer) Enzymes 0.0001-0.1%   Perfume0.1-0.5%  Water 5-10

[0209] 3) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant0.5-2.0% Sodium disilicate 25-40% Sodium citrate 30-55% Sodium carbonate 0-29% Sodium bicarbonate  0-20% Sodium perborate monohydrate  0-15%Tetraacetyl ethylene diamine (TAED) 0-6% Maleic acid/acrylic 0-5% acidcopolymer Clay 1-3% Polyamino acids  0-20% Sodium polyacrylate 0-8%Enzymes 0.0001-0.1%  

[0210] 4) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-2% Zeolite MAP 15-42% Sodium disilicate 30-34% Sodium citrate  0-12%Sodium carbonate  0-20% Sodium perborate monohydrate  7-15% Tetraacetylethylene 0-3% diamine (TAED) Polymer 0-4% Maleic acid/acrylic acidcopolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%  Sodium sulphate Balance

[0211] 5) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate12-20% Monopersulphate (2 KHSO₅.KHSO₄.K₂SO₄) 15-21% Bleach stabilizer0.1-2%   Maleic add/acrylic acid copolymer 0-6% Diethylene triaminepentaacetate,   0-2.5% pentasodium salt Enzymes 0.0001-0.1%   Sodiumsulphate, water Balance

[0212] 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANINGSURFACTANT SYSTEM Nonionic surfactant   0-1.5% Octadecyl dimethylamineN-oxide dihydrate 0-5% 80:20 wt.C18/C16 blend of octadecyl dimethylamine0-4% N-oxide dihydrate and hexadecyldimethyl amine N- oxide dihydrate70:30 wt.C18/C16 blend of octadecyl bis 0-5% (hydroxyethyl)amine N-oxideanhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrousC₁₃-C₁₅ alkyl ethoxysulfate with an average degree of  0-10%ethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfate with an average degree of0-5% ethoxylation of 3 C₁₃-C₁₅ ethoxylated alcohol with an averagedegree of 0-5% ethoxylation of 12 A blend of C₁₂-C₁₅ ethoxylatedalcohols with an   0-6.5% average degree of ethoxylation of 9 A blend ofC₁₃-C₁₅ ethoxylated alcohols with an 0-4% average degree of ethoxylationof 30 Sodium disilicate  0-33% Sodium tripolyphosphate  0-46% Sodiumcitrate  0-28% Citric acid  0-29% Sodium carbonate  0-20% Sodiumperborate monohydrate   0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4%Maleic add/acrylic acid copolymer   0-7.5% Sodium sulphate   0-12.5%Enzymes 0.0001-0.1%  

[0213] 7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION Liquidnonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0% Alkali metalsilicate 3.0-15.0% Alkali metal phosphate 20.0-40.0%  Liquid carrierselected from higher 25.0-45.0%  glycols, polyglycols, polyoxides,glycolethers Stabilizer (e.g. a partial ester of phosphoric acid and a0.5-7.0%  C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone)  0-1.5%Enzymes 0.0001-0.1%  

[0214] 8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionicsurfactant (e.g. alcohol ethoxylates) 2.0-10.0% Sodium silicate3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate 0.0-1.5% Stabilizing system (e.g. mixtures of finely divided 0.5-7.0%  siliconeand low molecular weight dialkyl polyglycol ethers) Low molecule weightpolyacrylate polymer 5.0-15.0% Clay gel thickener (e.g. bentonite)0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6%  Enzymes0.0001-0.1%   Liquid carrier selected from higher lycols, polyglycols,Balance polyoxides and glycol ethers

[0215] 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION C₁₂-C₁₄fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0%  Sodium citrate0-12%  Sodium tripolyphosphate 0-15%  Sodium carbonate 0-8%   Aluminiumtristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylatethickener 1.32-2.5%   Sodium polyacrylate 2.4-6.0%   Boric acid 0-4.0%Sodium formate  0-0.45% Calcium formate 0-0.2% Sodium n-decydiphenyloxide disulphonate 0-4.0% Monoethanol amine (MEA)  0-1.86% Sodiumhydroxide (50%) 1.9-9.3%   1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1%   Suds suppressor, dye, perfumes, water Balance

[0216] 10) LIQUID AUTOMATIC DISHWASHING COMPOSITION Alcohol ethoxylate0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20%Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate18-33%  Sodium citrate dihydrate 18-33%  Sodium stearate  0-2.5% Sodiumperborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED) 0-8% Maleic add/acrylic acid copolymer 4-8%  Enzymes 0.0001-0.1%  

[0217] 11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTEDBLEACH PARTICLES Sodium silicate  5-10% Tetrapotassium pyrophosphate 15-25% Sodium triphosphate  0-2% Potassium carbonate  4-8% Protectedbleach particles, e.g. chlorine  5-10% Polymeric thickener  0.7-1.5%Potassium hydroxide  0-2% Enzymes 0.0001-0.1 % Water Balance

[0218] 11) Automatic dishwashing compositions as described in 1), 2),3), 4), 6) and 10), wherein perborate is replaced by percarbonate.

[0219] 12) Automatic dishwashing compositions as described in 1)-6)which additionally contain a manganese catalyst. The manganese catalystmay, e.g., be one of the compounds described in “Efficient manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

Uses

[0220] The present invention is also directed to methods for using analpha-amylase variant of the invention in detergents, in particularlaundry detergent compositions and dishwashing detergent compositions,hard surface cleaning compositions, and in composition for desizing oftextiles, fabrics or garments, for production of pulp and paper, beermaking, ethanol production, and starch conversion processes as describedabove.

[0221] The present invention is further described by the followingexamples, which should not be construed as limiting the scope of theinvention.

Materials & Methods Enzymes

[0222] KSM-K36: SEQ ID NO: 2, disclosed in EP 1,022,334 deposited asFERM BP 6945.

[0223] KSM-K38: SEQ ID NO: 4, disclosed in EP 1,022,334, deposited asFERM BP-6946.

[0224]Bacillus subtilis SHA273: Protease and amylase deleted Bacillussubtilis strain (disclosed in WO 95/10603).

Detergent

[0225] Model detergent: A/P (Asia/Pacific) Model Detergent has thefollowing composition: 20% STPP (sodium tripolyphosphate), 25% Na₂SO₄,15% Na₂CO₃, 20% LAS (linear alkylbenzene sulfonate, Nansa 80S), 5%C₁₂-C₁₅ alcohol ethoxylate (Dobanol 25-7), 5% Na₂Si₂O₅, 0.3% NaCl.

[0226] Omo™ Multi Acao (Brazil),

[0227] Omo™ concentrated powder (EU) (Unilever)

[0228] Ariel Futur™ liquid (EU) (Procter and Gamble)

[0229] Commercial detergents containing alpha-amylase was inactivated bymicrowaves before wash.

Plasmids

[0230] pTVB110 is a plasmid replicating in Bacillus subtilis by the useof origin of replication from pUB110 (Gryczan, T. J. (1978), J. Bact.134:318-329). The plasmid further encodes the cat gene, conferringresistance towards chlorampenicol, obtained from plasmid pC194(Horinouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). Theplasmid harbors a truncated version of the Bacillus licheniformisalpha-amylase gene, amyL, such that the amyL promoter, signal sequenceand transcription terminator are present, but the plasmid does notprovide an amy-plus phenotype (halo formation on starch containingagar).

[0231] The alpha-amylase genes homologous to the KSM-K36 (SEQ ID NO: 1)and KSM-K38 (SEQ ID NO: 3) were cloned into the Pst1-Sal1 sites ofpTVB110. The coding amylase gene was obtained by PCR reaction usingpurified genomic DNA from the Bacillus KSM-K36 strain as template andthe DAX-8N (SEQ ID NO: 9) and DAX8C (SEQ ID NO: 10) primers.

Methods: General Molecular Biology Methods

[0232] Unless otherwise mentioned the DNA manipulations andtransformations were performed using standard methods of molecularbiology (Sambrook et al. (1989); Ausubel et al. (1995); Harwood andCutting (1990).

Filter Screening Assay

[0233] The assay can be used to screening of alpha-amylase variantshaving an improved stability at high pH compared to the parent enzymeand alpha-amylase variants having an improved stability at high pH andmedium temperatures compared to the parent enzyme depending of thescreening temperature setting.

High pH Filter Assay

[0234] Bacillus libraries are plated on a sandwich of cellulose acetate(OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulosefilters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TYagar plates with 10 micro g/ml kanamycin at 37° C. for at least 21hours. The cellulose acetate layer is located on the TY agar plate.

[0235] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter and the nitrocellulose filter with bound variantsis transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 andincubated at room temperature (can be altered from 10°-60° C.) for 15min. The cellulose acetate filters with colonies are stored on theTY-plates at room temperature until use. After incubation, residualactivity is detected on plates containing 1% agarose, 0.2% starch inglycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours. at room temperature. After removal of the filters the assayplates are stained with 10% Lugol solution. Starch degrading variantsare detected as white spots on dark blue background and then identifiedon the storage plates. Positive variants are rescreened twice under thesame conditions as the first screen.

Low Calcium Filter Assay

[0236] The Bacillus library are plated on a sandwich of celluloseacetate (OE 67, Schleicher & Schuell, Dassel, Germany)—andnitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel,Germany) on TY agar plates with a relevant antibiotic, e.g., kanamycinor chloramphenicol, at 37° C. for at least 21 hours. The celluloseacetate layer is located on the TY agar plate.

[0237] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter and the nitrocellulose filter with bound variantsis transferred to a container with carbonate/bicarbonate buffer pH8.5-10 and with different EDTA concentrations (0.001 mM -100 mM). Thefilters are incubated at room temperature for 1 hour. The celluloseacetate filters with colonies are stored on the TY-plates at roomtemperature until use. After incubation, residual activity is detectedon plates containing 1% agarose, 0.2% starch in carbonatelbicarbonatebuffer pH 8.5-10. The assay plates with nitrocellulose filters aremarked the same way as the filter sandwich and incubated for 2 hours atroom temperature. After removal of the filters the assay plates arestained with 10% Lugol solution. Starch degrading variants are detectedas white spots on dark blue background and then identified on thestorage plates. Positive variants are rescreened twice under the sameconditions as the first screen.

Determination of Isoelectric Point

[0238] The pI is determined by isoelectric focusing (ex: Pharmacia,Ampholine, pH 3.5-9.3).

Fermentation of Alpha-Amylases and Variants

[0239] Fermentation may be performed by methods well known in the art oras follows.

[0240] A B. subtilis strain harboring the relevant expression plasmid isstreaked on a LB-agar plate with a relevant antibiotic, and grownovernight at 37° C. The colonies are transferred to 100 ml BPX mediasupplemented with a relevant antibiotic (for instance 10 mg/lchloroamphinicol) in a 500 ml shaking flask.

Composition of BPX Medium

[0241] Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/lSodium caseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O 9 g/lPluronic ™ 0.1 g/l

[0242] The culture is shaken at 37° C. at 270 rpm for 4 to 5 days.

[0243] Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on an UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2 MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions, which contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

Stability Determination

[0244] The amylase stability is measured using the method as follows:

[0245] The enzyme is incubated under the relevant conditions. Samplesare taken at various time points, e.g., after 0, 5, 10, 15 and 30minutes and diluted 25 times (same dilution for all taken samples) inassay buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity ismeasured using the Phadebas assay (Pharmacia) under standard conditionspH 7.3, 37° C.

[0246] The activity measured before incubation (0 minutes) is used asreference (100%). The decline in percent is calculated as a function ofthe incubation time. The table shows the residual activity after, e.g.,30 minutes of incubation.

Measurement of the Calcium- and pH-Dependent Stability

[0247] Normally industrial liquefaction processes runs using pH 6.0-6.2as liquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95° C.-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

[0248] 1. lower pH than pH 6.2 and/or

[0249] 2. at free calcium levels lower than 40 ppm free calcium.

[0250] Two different methods can be used to measure the alterations instability obtained by the different substitutions in the alpha-amylasein question:

[0251] Method 1. One assay which measures the stability at reduced pH,pH 5.0, in the presence of 5 ppm free calcium.

[0252] 10 micro g of the variant are incubated under the followingconditions: A 0.1 M acetate solution, pH adjusted to pH 5.0, containing5 ppm calcium and 5% w/w common corn starch (free of calcium).Incubation is made in a water bath at 95° C. for 30 minutes.

[0253] Method 2. One assay, which measure the stability in the absenceof free calcium and where the pH is maintained at pH 6.0. This assaymeasures the decrease in calcium sensitivity:

[0254] 10 micro g of the variant were incubated under the followingconditions: A 0.1 M acetate solution, pH adjusted to pH 6.0, containing5% w/w common corn starch (free of calcium). Incubation was made in awater bath at 95° C. for 30 minutes.

Assays for Alpha-Amylase Activity 1. Phadebas Assay

[0255] Alpha-amylase activity is determined by a method employingPhadebas) tablets as substrate. Phadebas tablets (Phadebas® AmylaseTest, supplied by Pharmacia Diagnostic) contain a cross-linked insolubleblue-colored starch polymer, which has been mixed with bovine serumalbumin and a buffer substance and tabletted.

[0256] For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

[0257] It is important that the measured 620 nm absorbance after 10 or15 minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

2. Alternative Method

[0258] Alpha-amylase activity is determined by a method employing thePNP-G7 substrate. PNP-G7 which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be cleaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at λ=405 nm. (400-420 nm.). Kits containingPNP-G7 substrate and alpha-Glucosidase is manufactured byBoehringer-Mannheim (cat. No. 1054635).

[0259] To prepare the substrate one bottle of substrate (BM 1442309) isadded to 5 ml buffer (BM1442309). To prepare the alpha-glucosidase onebottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer(BM1442309). The working solution is made by mixing 5 mlalpha-Glucosidase solution with 0.5 ml substrate.

[0260] The assay is performed by transforming 20 micro I enzyme solutionto a 96 well microtitre plate and incubating at 25° C. 200 micro Iworking solution, 25° C. is added. The solution is mixed andpre-incubated 1 minute and absorption is measured every 15 secounds over3 minutes at OD 405 nm.

[0261] The slope of the time dependent absorption-curve is directlyproportional to the specific activity (activity per mg enzyme) of thealpha-amylase in question under the given set of conditions.

Specific Activity Determination

[0262] The specific activity is determined as activity/mg enzyme usingone of the methods described above. The manufactures instructions arefollowed (see also below under “Assay for alpha-amylase activity).

Oxidation Stability Determination

[0263] Raw filtered culture broths with different vatiants of theinvention are diluted to an amylase activity of 100 KNU/ml (definedabove) in 50 mM of a Britton-Robinson buffer at pH 9.0 and incubated at40° C. Subsequently H₂O₂ is added to a concentration of 200 mM, and thepH value is re-adjusted to 9.0. The activity is now measured after 15seconds and after 5, 15, and 30 minutes. The absorbance of the resultingblue solution, measured spectrophotometrically at 620 nm, is a functionof the alpha-amylase activity.

Washing Performance

[0264] Washing performance is evaluated by washing soiled test swatchesfor 15 and 30 minutes at 25° C. and 40° C., respectively; at a pH in therange from 9-10.5; water hardness in the range from 6 to 15□dH; Ca:Mgratio of from 2:1 to 4:1, in different detergent solutions (see above asdescribed above in the Materials section) dosed from 3 to 5 g/ldependent on the detergent with the alpha-amylase variant in question.

[0265] The recombinant alpha-amylase variant is added to the detergentsolutions at concentrations of for instance 0.01-5 mg/l. The testswatches aree soiled with orange rice starch (CS-28 swatches availablefrom CFT, Center for Test Material, Holland).

[0266] After washing, the swatches are evaluated by measuring theremission at 460 nm using an Elrepho Remission Spectrophotometer. Theresults are expressed as ΔR=remission of the swatch washed with thealpha-amylase minus the remission of a swatch washed at the sameconditions without the alpha-amylase.

General Method for Random Mutagenesis by Use of the DOPE Program

[0267] The random mutagenesis may be carried out as follows:

[0268] 1. Select regions of interest for modification in the parentenzyme

[0269] 2. Decide on mutation sites and non-mutated sites in the selectedregion

[0270] 3. Decide on which kind of mutations should be carried out, e.g.with respect to the desired stability and/or performance of the variantto be constructed

[0271] 4. Select structurally reasonable mutations.

[0272] 5. Adjust the residues selected by step 3 with regard to step 4.

[0273] 6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

[0274] 7. If necessary, adjust the wanted residues to genetic coderealism (e.g., taking into account constraints resulting from thegenetic code (e.g. in order to avoid introduction of stop codons))(theskilled person will be aware that some codon combinations cannot be usedin practice and will need to be adapted)

[0275] 8. Make primers

[0276] 9. Perform random mutagenesis by use of the primers

[0277] 10. Select resulting α-amylase variants by screening for thedesired improved properties.

[0278] Suitable dope algorithms for use in step 6 are well known in theart. One algorithm is described by Tomandl, D. et al., Journal ofComputer-Aided Molecular Design, 11 (1997), pp. 29-38). Anotheralgorithm, DOPE, is described in the following:

The Dope Program

[0279] The “DOPE” program is a computer algorithm useful to optimize thenucleotide composition of a codon triplet in such a way that it encodesan amino acid distribution which resembles most the wanted amino aciddistribution. In order to assess which of the possible distributions isthe most similar to the wanted amino acid distribution, a scoringfunction is needed. In the “Dope” program the following function wasfound to be suited:${s \equiv {\prod\limits_{i = 1}^{N}\left( {\frac{x_{i}^{y_{i}}}{y_{i}^{y_{i}}}\frac{\left( {1 - x_{i}} \right)^{1 - y_{i}}}{\left( {1 - y_{i}} \right)^{1 - y_{i}}}} \right)^{w_{i}}}},$

[0280] where the x_(i)'s are the obtained amounts of amino acids andgroups of amino acids as calculated by the program, y_(i)'s are thewanted amounts of amino acids and groups of amino acids as defined bythe user of the program (e.g. specify which of the 20 amino acids orstop codons are wanted to be introduced, e.g. with a certain percentage(e.g. 90% Ala, 3% Ile, 7% Val), and w_(i)'s are assigned weight factorsas defined by the user of the program (e.g., depending on the importanceof having a specific amino acid residue inserted into the position inquestion). N is 21 plus the number of amino acid groups as defined bythe user of the program. For purposes of this function 0° is defined asbeing 1.

[0281] A Monte-Carlo algorithm (one example being the one described byValleau, J. P. & Whittington, S. G. (1977) A guide to Mont Carlo forstatistical mechanics: 1 Highways. In “Stastistical Mechanics, Part A”Equlibrium Techniqeues ed. B. J. Berne, New York:

[0282] Plenum) is used for finding the maximum value of this function.In each iteration the following steps are performed:

[0283] 1. A new random nucleotide composition is chosen for each base,where the absolute difference between the current and the newcomposition is smaller than or equal to d for each of the fournucleotides G,A,T,C in all three positions of the codon (see below fordefinition of d).

[0284] 2. The scores of the new composition and the current compositionare compared by the use of the function s as described above. If the newscore is higher or equal to the score of the current composition, thenew composition is kept and the current composition is changed to thenew one. If the new score is smaller, the probability of keeping the newcomposition is exp(1000(new_score-current_score)).

[0285] A cycle normally consists of 1000 iterations as described abovein which d is decreasing linearly from 1 to 0. One hundred or morecycles are performed in an optimization process. The nucleotidecomposition resulting in the highest score is finally presented.

EXAMPLES Example 1 Construction of Stabilised Amylase Variants

[0286] Stabilising amino acid substitutions can be introduced by themega-primer-PCR method described by Sarkar and Sommer, 1990,BioTechniques 8: 404-407, using a mutagenesis primer and two specificprimers binding upstreams and down-streams, respectively of both thepoint of mutation and the restriction sites to be used for cloning.

[0287] To introduce the substitutions: E84Q, N96D, A315S, A445V, G464N,N121D and N393H the following mutagenesis primers could be used: PrimerAmrk752-E84Q: ctaaggcacagctt caa cgagctattgggtcc (SEQ ID NO:11) PrimerAmrk752-N96D: ccttaaatctaatgatatc gat gtatacggagatg (SEQ ID NO:12)Primer Amrk752-A315S: ttataatttttaccgg tct tcacaacaaggtgga (SEQ IDNO:13) Primer Amrk752-A445V: gtaggacgtcagaat gta ggacaaacatggac (SEQ IDNO:14) Primer Amrk752-G464N: ccgttacaattaat aac gatggatggggcgaattc (SEQID NO:15) Primer Amrk23O-N121D: gcaagctgttcaagta gat ccaacgaatcgttgg(SEQ ID NO:16) Primer Amrk230-N390H: gcttgatgcacgtcaa gattacgcatatggcacg (SEQ ID NO:17) —where the mutated codon is highlighted.

[0288] The amylase variant Amrk752 can be constructed by simultaneousintroduction of the first five substitutions into SEQ 4 while Amrk230can be constructed by introducing the last to substitutions into SEQ 4.

[0289] In a similar manner can Amrk299 be constructed on the basis ofSEQ 4 by introducing the substitutions: T125S, S144P, I173L, D210E,N393H, V408I, R442Q, N444H, Q448A and G464S.

[0290] Wild type and variant amylases could be expressed in B.subtlisstrains deficient of background amylase and protease activity andfollowing be purified by conventional purifications methods.

Example 2 Activity at Alkaline pH

[0291] The relative activity of the amylases at alkaline pH was measuredon culture broth, and the activity at pH 8 was defined to be 100% forcomparison of the results in the table below. The Phadebas amylase assaysystem manufacted by Pharmacia AB was used in pH 10 buffer at 50° C. andwith 15 min reaction time. Amylase pH 8 pH 10 SEQ 4 100% 6% Amrk230 100%94% Amrk299 100% 19% Amrk752 100% 49%

[0292]

1 17 1 1650 DNA Bacillus sp. CDS (65)..(1567) sig_peptide (65)..(128)mat_peptide (128)..() 1 cttgaatcat tatttaaagc tggttatgat atatgtaagcgttatcatta aaaggaggta 60 tttg atg aaa aga tgg gta gta gca atg ctg gcagtg tta ttt tta ttt 109 Met Lys Arg Trp Val Val Ala Met Leu Ala Val LeuPhe Leu Phe -20 -15 -10 cct tcg gta gta gtt gca gat ggc ttg aat gga acgatg atg cag tat 157 Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr MetMet Gln Tyr -5 -1 1 5 10 tat gag tgg cat cta gag aat gat ggg caa cac tggaat cgg ttg cat 205 Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp AsnArg Leu His 15 20 25 gat gat gcc gaa gct tta agt aat gcg ggt att aca gctatt tgg ata 253 Asp Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala IleTrp Ile 30 35 40 ccc cca gcc tac aaa gga aat agt cag gct gat gtt ggg tatggt gca 301 Pro Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr GlyAla 45 50 55 tac gac ctt tat gat tta ggg gag ttt aat caa aaa ggt acc gttcga 349 Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg60 65 70 acg aaa tac ggg aca aag gct cag ctt gag cga gct ata ggg tcc cta397 Thr Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu 7580 85 90 aag tcg aat gat atc aat gtt tat ggg gat gtc gta atg aat cat aaa445 Lys Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys 95100 105 tta gga gct gat ttc acg gag gca gtg caa gct gtt caa gta aat cct493 Leu Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro 110115 120 tcg aac cgt tgg cag gat att tca ggt gtc tac acg att gat gca tgg541 Ser Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp 125130 135 acg gga ttt gac ttt cca ggg cgc aac aat gcc tat tcc gat ttt aaa589 Thr Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys 140145 150 tgg aga tgg ttc cat ttt aat ggc gtt gac tgg gat caa cgc tat caa637 Trp Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln 155160 165 170 gaa aac cat ctt ttt cgc ttt gca aat acg aac tgg aac tgg cgagtg 685 Glu Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val175 180 185 gat gaa gag aat ggt aat tat gac tat tta tta gga tcg aac attgac 733 Asp Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp190 195 200 ttt agc cac cca gag gtt caa gag gaa tta aag gat tgg ggg agctgg 781 Phe Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp205 210 215 ttt acg gat gag cta gat tta gat ggg tat cga ttg gat gct attaag 829 Phe Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys220 225 230 cat att cca ttc tgg tat acg tca gat tgg gtt agg cat cag cgaagt 877 His Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser235 240 245 250 gaa gca gac caa gat tta ttt gtc gta ggg gag tat tgg aaggat gac 925 Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys AspAsp 255 260 265 gta ggt gct ctc gaa ttt tat tta gat gaa atg aat tgg gagatg tct 973 Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu MetSer 270 275 280 cta ttc gat gtt ccg ctc aat tat aat ttt tac cgg gct tcaaag caa 1021 Leu Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser LysGln 285 290 295 ggc gga agc tat gat atg cgt aat att tta cga gga tct ttagta gaa 1069 Gly Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu ValGlu 300 305 310 gca cat ccg att cat gca gtt acg ttt gtt gat aat cat gatact cag 1117 Ala His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp ThrGln 315 320 325 330 cca gga gag tca tta gaa tca tgg gtc gct gat tgg tttaag cca ctt 1165 Pro Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe LysPro Leu 335 340 345 gct tat gcg aca atc ttg acg cgt gaa ggt ggt tat ccaaat gta ttt 1213 Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro AsnVal Phe 350 355 360 tac ggt gac tac tat ggg att cct aac gat aac att tcagct aag aag 1261 Tyr Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser AlaLys Lys 365 370 375 gat atg att gat gag ttg ctt gat gca cgt caa aat tacgca tat ggc 1309 Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr AlaTyr Gly 380 385 390 aca caa cat gac tat ttt gat cat tgg gat atc gtt ggatgg aca aga 1357 Thr Gln His Asp Tyr Phe Asp His Trp Asp Ile Val Gly TrpThr Arg 395 400 405 410 gaa ggt aca tcc tca cgt cct aat tcg ggt ctt gctact att atg tcc 1405 Glu Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala ThrIle Met Ser 415 420 425 aat ggt cct gga gga tca aaa tgg atg tac gta ggacag caa cat gca 1453 Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly GlnGln His Ala 430 435 440 gga caa acg tgg aca gat tta act ggc aat cac gcggcg tcg gtt acg 1501 Gly Gln Thr Trp Thr Asp Leu Thr Gly Asn His Ala AlaSer Val Thr 445 450 455 att aat ggt gat ggc tgg ggc gaa ttc ttt aca aatgga gga tct gta 1549 Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn GlyGly Ser Val 460 465 470 tcc gtg tat gtg aac caa taataaaaag ccttgagaagggattcctcc 1597 Ser Val Tyr Val Asn Gln 475 480 ctaactcaag gctttctttatgtcgtttag ctcaacgctt ctacgaagct tta 1650 2 501 PRT Bacillus sp. 2 MetLys Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20 -15 -10Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr -5 -1 15 10 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 1520 25 Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro 3035 40 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 4550 55 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 6065 70 75 Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys80 85 90 Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Leu95 100 105 Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn ProSer 110 115 120 Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp AlaTrp Thr 125 130 135 Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser AspPhe Lys Trp 140 145 150 155 Arg Trp Phe His Phe Asn Gly Val Asp Trp AspGln Arg Tyr Gln Glu 160 165 170 Asn His Leu Phe Arg Phe Ala Asn Thr AsnTrp Asn Trp Arg Val Asp 175 180 185 Glu Glu Asn Gly Asn Tyr Asp Tyr LeuLeu Gly Ser Asn Ile Asp Phe 190 195 200 Ser His Pro Glu Val Gln Glu GluLeu Lys Asp Trp Gly Ser Trp Phe 205 210 215 Thr Asp Glu Leu Asp Leu AspGly Tyr Arg Leu Asp Ala Ile Lys His 220 225 230 235 Ile Pro Phe Trp TyrThr Ser Asp Trp Val Arg His Gln Arg Ser Glu 240 245 250 Ala Asp Gln AspLeu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 255 260 265 Gly Ala LeuGlu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 270 275 280 Phe AspVal Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln Gly 285 290 295 GlySer Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 300 305 310315 His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 320325 330 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala335 340 345 Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val PheTyr 350 355 360 Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala LysLys Asp 365 370 375 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr AlaTyr Gly Thr 380 385 390 395 Gln His Asp Tyr Phe Asp His Trp Asp Ile ValGly Trp Thr Arg Glu 400 405 410 Gly Thr Ser Ser Arg Pro Asn Ser Gly LeuAla Thr Ile Met Ser Asn 415 420 425 Gly Pro Gly Gly Ser Lys Trp Met TyrVal Gly Gln Gln His Ala Gly 430 435 440 Gln Thr Trp Thr Asp Leu Thr GlyAsn His Ala Ala Ser Val Thr Ile 445 450 455 Asn Gly Asp Gly Trp Gly GluPhe Phe Thr Asn Gly Gly Ser Val Ser 460 465 470 475 Val Tyr Val Asn Gln480 3 1745 DNA Bacillus CDS (190)..(1692) sig_peptide (190)..(253)mat_peptide (253)..() 3 aactaagtaa catcgattca ggataaaagt atgcgaaacgatgcgcaaaa ctgcgcaact 60 actagcactc ttcagggact aaaccacctt ttttccaaaaatgacatcat ataaacaaat 120 ttgtctacca atcactattt aaagctgttt atgatatatgtaagcgttat cattaaaagg 180 aggtatttg atg aga aga tgg gta gta gca atg ttggca gtg tta ttt tta 231 Met Arg Arg Trp Val Val Ala Met Leu Ala Val LeuPhe Leu -20 -15 -10 ttt cct tcg gta gta gtt gca gat gga ttg aac ggt acgatg atg cag 279 Phe Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr MetMet Gln -5 -1 1 5 tat tat gag tgg cat ttg gaa aac gac ggg cag cat tggaat cgg ttg 327 Tyr Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp AsnArg Leu 10 15 20 25 cac gat gat gcc gca gct ttg agt gat gct ggt att acagct att tgg 375 His Asp Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr AlaIle Trp 30 35 40 att ccg cca gcc tac aaa ggt aat agt cag gcg gat gtt gggtac ggt 423 Ile Pro Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly TyrGly 45 50 55 gca tac gat ctt tat gat tta gga gag ttc aat caa aag ggt actgtt 471 Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val60 65 70 cga acg aaa tac gga act aag gca cag ctt gaa cga gct att ggg tcc519 Arg Thr Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser 7580 85 ctt aaa tct aat gat atc aat gta tac gga gat gtc gtg atg aat cat567 Leu Lys Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His 9095 100 105 aaa atg gga gct gat ttt acg gag gca gtg caa gct gtt caa gtaaat 615 Lys Met Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn110 115 120 cca acg aat cgt tgg cag gat att tca ggt gcc tac acg att gatgcg 663 Pro Thr Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala125 130 135 tgg acg ggt ttc gac ttt tca ggg cgt aac aac gcc tat tca gatttt 711 Trp Thr Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe140 145 150 aag tgg aga tgg ttc cat ttt aat ggt gtt gac tgg gat cag cgctat 759 Lys Trp Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr155 160 165 caa gaa aat cat att ttc cgc ttt gca aat acg aac tgg aac tggcga 807 Gln Glu Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg170 175 180 185 gtg gat gaa gag aac ggt aat tat gat tac ctg tta gga tcgaat atc 855 Val Asp Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser AsnIle 190 195 200 gac ttt agt cat cca gaa gta caa gat gag ttg aag gat tggggt agc 903 Asp Phe Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp GlySer 205 210 215 tgg ttt acc gat gag tta gat ttg gat ggt tat cgt tta gatgct att 951 Trp Phe Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp AlaIle 220 225 230 aaa cat att cca ttc tgg tat aca tct gat tgg gtt cgg catcag cgc 999 Lys His Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His GlnArg 235 240 245 aac gaa gca gat caa gat tta ttt gtc gta ggg gaa tat tggaag gat 1047 Asn Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp LysAsp 250 255 260 265 gac gta ggt gct ctc gaa ttt tat tta gat gaa atg aattgg gag atg 1095 Asp Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn TrpGlu Met 270 275 280 tct cta ttc gat gtt cca ctt aat tat aat ttt tac cgggct tca caa 1143 Ser Leu Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg AlaSer Gln 285 290 295 caa ggt gga agc tat gat atg cgt aat att tta cga ggatct tta gta 1191 Gln Gly Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly SerLeu Val 300 305 310 gaa gcg cat ccg atg cat gca gtt acg ttt gtt gat aatcat gat act 1239 Glu Ala His Pro Met His Ala Val Thr Phe Val Asp Asn HisAsp Thr 315 320 325 cag cca ggg gag tca tta gag tca tgg gtt gct gat tggttt aag cca 1287 Gln Pro Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp PheLys Pro 330 335 340 345 ctt gct tat gcg aca att ttg acg cgt gaa ggt ggttat cca aat gta 1335 Leu Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly TyrPro Asn Val 350 355 360 ttt tac ggt gat tac tat ggg att cct aac gat aacatt tca gct aaa 1383 Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn IleSer Ala Lys 365 370 375 aaa gat atg att gat gag ctg ctt gat gca cgt caaaat tac gca tat 1431 Lys Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln AsnTyr Ala Tyr 380 385 390 ggc acg cag cat gac tat ttt gat cat tgg gat gttgta gga tgg act 1479 Gly Thr Gln His Asp Tyr Phe Asp His Trp Asp Val ValGly Trp Thr 395 400 405 agg gaa gga tct tcc tcc aga cct aat tca ggc cttgcg act att atg 1527 Arg Glu Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu AlaThr Ile Met 410 415 420 425 tcg aat gga cct ggt ggt tcc aag tgg atg tatgta gga cgt cag aat 1575 Ser Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr ValGly Arg Gln Asn 430 435 440 gca gga caa aca tgg aca gat tta act ggt aataac gga gcg tcc gtt 1623 Ala Gly Gln Thr Trp Thr Asp Leu Thr Gly Asn AsnGly Ala Ser Val 445 450 455 aca att aat ggc gat gga tgg ggc gaa ttc tttacg aat gga gga tct 1671 Thr Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe ThrAsn Gly Gly Ser 460 465 470 gta tcc gtg tac gtg aac caa taacaaaaagccttgagaag ggattcctcc 1722 Val Ser Val Tyr Val Asn Gln 475 480ctaactcaag gctttcttta tgt 1745 4 501 PRT Bacillus 4 Met Arg Arg Trp ValVal Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20 -15 -10 Ser Val Val ValAla Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr -5 -1 1 5 10 Glu Trp HisLeu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 15 20 25 Asp Ala AlaAla Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 30 35 40 Pro Ala TyrLys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 45 50 55 Asp Leu TyrAsp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 60 65 70 75 Lys TyrGly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 80 85 90 Ser AsnAsp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 95 100 105 GlyAla Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 110 115 120Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 125 130135 Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp 140145 150 155 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr GlnGlu 160 165 170 Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp ArgVal Asp 175 180 185 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser AsnIle Asp Phe 190 195 200 Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp TrpGly Ser Trp Phe 205 210 215 Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg LeuAsp Ala Ile Lys His 220 225 230 235 Ile Pro Phe Trp Tyr Thr Ser Asp TrpVal Arg His Gln Arg Asn Glu 240 245 250 Ala Asp Gln Asp Leu Phe Val ValGly Glu Tyr Trp Lys Asp Asp Val 255 260 265 Gly Ala Leu Glu Phe Tyr LeuAsp Glu Met Asn Trp Glu Met Ser Leu 270 275 280 Phe Asp Val Pro Leu AsnTyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 285 290 295 Gly Ser Tyr Asp MetArg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 300 305 310 315 His Pro MetHis Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 320 325 330 Gly GluSer Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 335 340 345 TyrAla Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 350 355 360Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 365 370375 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr 380385 390 395 Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr ArgGlu 400 405 410 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile MetSer Asn 415 420 425 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg GlnAsn Ala Gly 430 435 440 Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly AlaSer Val Thr Ile 445 450 455 Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr AsnGly Gly Ser Val Ser 460 465 470 475 Val Tyr Val Asn Gln 480 5 1920 DNABacillus licheniformis CDS (421)..(1872) 5 cggaagattg gaagtacaaaaataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtcttaatgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaagcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggctattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatatagggaaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaaggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatttgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acgctg atg cag tat ttt gaa tgg tac atg ccc 468 Ala Asn Leu Asn Gly Thr LeuMet Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 aat gac ggc caa cat tggagg cgt ttg caa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 gct gaa cac ggt att act gccgtc tgg att ccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala ValTrp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tacggt gct tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gttcgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val ArgThr Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agtctt cat tcc cgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser LeuHis Ser Arg Asp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaaggc ggc gct gat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys GlyGly Ala Asp Ala Thr 100 105 110 gaa gat gta acc gcg gtt gaa gtc gat cccgct gac cgc aac cgc gta 804 Glu Asp Val Thr Ala Val Glu Val Asp Pro AlaAsp Arg Asn Arg Val 115 120 125 att tca gga gaa cac cta att aaa gcc tggaca cat ttt cat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp ThrHis Phe His Phe Pro 130 135 140 ggg cgc ggc agc aca tac agc gat ttt aaatgg cat tgg tac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys TrpHis Trp Tyr His Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cgaaag ctg aac cgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg LysLeu Asn Arg Ile Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaagtt tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu ValSer Asn Glu Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atcgat tat gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile AspTyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc acttgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp Gly Thr TrpTyr Ala Asn Glu Leu Gln 210 215 220 ttg gac ggt ttc cgt ctt gat gct gtcaaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys Phe Ser Phe 225 230 235 240 ttg cgg gat tgg gtt aat cat gtcagg gaa aaa acg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val ArgGlu Lys Thr Gly Lys Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cagaat gac ttg ggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln AsnAsp Leu Gly Ala Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aatcat tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn HisSer Val Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcgaca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser ThrGln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt acg gtcgtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 gtt aca ttt gtc gat aac catgat aca cag ccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His AspThr Gln Pro Gly Gln Ser Leu Glu 325 330 335 tcg act gtc caa aca tgg tttaag ccg ctt gct tac gct ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe LysPro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cctcag gtt ttc tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro GlnVal Phe Tyr Gly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgcgaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg GluIle Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg agaaaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg LysGln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 gat tat ttc gac cac catgac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His AspIle Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 agc tcg gtt gca aat tcaggt ttg gcg gca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser GlyLeu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atgtat gtc ggc cgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met TyrVal Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc ggaaac cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly AsnArg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag tttcac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe HisVal Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga tagaagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt1920 6 483 PRT Bacillus licheniformis 6 Ala Asn Leu Asn Gly Thr Leu MetGln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr AlaVal Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys GlyThr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser AlaIle Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val ValIle Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr AlaVal Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly GluHis Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg GlySer Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 AspGly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe SerPhe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr GlyLys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu GlyAla Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser ValPhe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr GlnGly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn HisAsp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr TrpPhe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser GlyTyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly AspSer Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro IleLeu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 AspTyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser IleTyr 465 470 475 480 Val Gln Arg 7 1776 DNA Bacillus sp. CDS(145)..(1692) sig_peptide (145)..(238) mat_peptide (238)..() 7atataaattt gaaatgaaca cctatgaaaa tatggtagcg attgcgcgac gagaaaaaac 60ttgggagtta ggaagtgata ttaaaggatt ttttttgact tgttgtgaaa acgcttgcat 120aaattgaagg agagggtgct tttt atg aaa ctt cat aac cgt ata att agc 171 MetLys Leu His Asn Arg Ile Ile Ser -30 -25 gta cta tta aca cta ttg tta gctgta gct gtt ttg ttt cca tat atg 219 Val Leu Leu Thr Leu Leu Leu Ala ValAla Val Leu Phe Pro Tyr Met -20 -15 -10 acg gaa cca gca caa gcc cat cataat ggg acg aat ggg acc atg atg 267 Thr Glu Pro Ala Gln Ala His His AsnGly Thr Asn Gly Thr Met Met -5 -1 1 5 10 cag tat ttt gaa tgg cat ttg ccaaat gac ggg aac cac tgg aac agg 315 Gln Tyr Phe Glu Trp His Leu Pro AsnAsp Gly Asn His Trp Asn Arg 15 20 25 tta cga gat gac gca gct aac tta aagagt aaa ggg att acc gct gtt 363 Leu Arg Asp Asp Ala Ala Asn Leu Lys SerLys Gly Ile Thr Ala Val 30 35 40 tgg att cct cct gca tgg aag ggg act tcgcaa aat gat gtt ggg tat 411 Trp Ile Pro Pro Ala Trp Lys Gly Thr Ser GlnAsn Asp Val Gly Tyr 45 50 55 ggt gcc tat gat ttg tac gat ctt ggt gag tttaac caa aag gga acc 459 Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe AsnGln Lys Gly Thr 60 65 70 gtc cgt aca aaa tat ggc aca agg agt cag ttg caaggt gcc gtg aca 507 Val Arg Thr Lys Tyr Gly Thr Arg Ser Gln Leu Gln GlyAla Val Thr 75 80 85 90 tct ttg aaa aat aac ggg att caa gtt tat ggg gatgtc gtg atg aat 555 Ser Leu Lys Asn Asn Gly Ile Gln Val Tyr Gly Asp ValVal Met Asn 95 100 105 cat aaa ggt gga gca gac ggg aca gag atg gta aatgcg gtg gaa gtg 603 His Lys Gly Gly Ala Asp Gly Thr Glu Met Val Asn AlaVal Glu Val 110 115 120 aac cga agc aac cga aac caa gaa ata tca ggt gaatac acc att gaa 651 Asn Arg Ser Asn Arg Asn Gln Glu Ile Ser Gly Glu TyrThr Ile Glu 125 130 135 gca tgg acg aaa ttt gat ttc cct gga aga gga aatacc cat tcc aac 699 Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly Asn ThrHis Ser Asn 140 145 150 ttt aaa tgg cgc tgg tat cat ttt gat ggg aca gattgg gat cag tca 747 Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Thr Asp TrpAsp Gln Ser 155 160 165 170 cgt cag ctt cag aac aaa ata tat aaa ttc agaggt acc gga aag gca 795 Arg Gln Leu Gln Asn Lys Ile Tyr Lys Phe Arg GlyThr Gly Lys Ala 175 180 185 tgg gac tgg gaa gta gat ata gag aac ggc aactat gat tac ctt atg 843 Trp Asp Trp Glu Val Asp Ile Glu Asn Gly Asn TyrAsp Tyr Leu Met 190 195 200 tat gca gac att gat atg gat cat cca gaa gtaatc aat gaa ctt aga 891 Tyr Ala Asp Ile Asp Met Asp His Pro Glu Val IleAsn Glu Leu Arg 205 210 215 aat tgg gga gtt tgg tat aca aat aca ctt aatcta gat gga ttt aga 939 Asn Trp Gly Val Trp Tyr Thr Asn Thr Leu Asn LeuAsp Gly Phe Arg 220 225 230 atc gat gct gtg aaa cat att aaa tac agc tatacg aga gat tgg cta 987 Ile Asp Ala Val Lys His Ile Lys Tyr Ser Tyr ThrArg Asp Trp Leu 235 240 245 250 aca cat gtg cgt aac acc aca ggt aaa ccaatg ttt gca gtt gca gaa 1035 Thr His Val Arg Asn Thr Thr Gly Lys Pro MetPhe Ala Val Ala Glu 255 260 265 ttt tgg aaa aat gac ctt gct gca atc gaaaac tat tta aat aaa aca 1083 Phe Trp Lys Asn Asp Leu Ala Ala Ile Glu AsnTyr Leu Asn Lys Thr 270 275 280 agt tgg aat cac tcc gtg ttc gat gtt cctctt cat tat aat ttg tac 1131 Ser Trp Asn His Ser Val Phe Asp Val Pro LeuHis Tyr Asn Leu Tyr 285 290 295 aat gca tct aat agt ggt ggc tat ttt gatatg aga aat att tta aat 1179 Asn Ala Ser Asn Ser Gly Gly Tyr Phe Asp MetArg Asn Ile Leu Asn 300 305 310 ggt tct gtc gta caa aaa cac cct ata catgca gtc aca ttt gtt gat 1227 Gly Ser Val Val Gln Lys His Pro Ile His AlaVal Thr Phe Val Asp 315 320 325 330 aac cat gac tct cag cca gga gaa gcattg gaa tcc ttt gtt caa tcg 1275 Asn His Asp Ser Gln Pro Gly Glu Ala LeuGlu Ser Phe Val Gln Ser 335 340 345 tgg ttc aaa cca ctg gca tat gca ttgatt ctg aca agg gag caa ggt 1323 Trp Phe Lys Pro Leu Ala Tyr Ala Leu IleLeu Thr Arg Glu Gln Gly 350 355 360 tac cct tcc gta ttt tac ggt gat tactac ggt ata cca act cat ggt 1371 Tyr Pro Ser Val Phe Tyr Gly Asp Tyr TyrGly Ile Pro Thr His Gly 365 370 375 gtt cct tcg atg aaa tct aaa att gatcca ctt ctg cag gca cgt caa 1419 Val Pro Ser Met Lys Ser Lys Ile Asp ProLeu Leu Gln Ala Arg Gln 380 385 390 acg tat gcc tac gga acc caa cat gattat ttt gat cat cat gat att 1467 Thr Tyr Ala Tyr Gly Thr Gln His Asp TyrPhe Asp His His Asp Ile 395 400 405 410 atc ggc tgg acg aga gaa ggg gacagc tcc cac cca aat tca gga ctt 1515 Ile Gly Trp Thr Arg Glu Gly Asp SerSer His Pro Asn Ser Gly Leu 415 420 425 gca act att atg tcc gat ggg ccaggg ggt aat aaa tgg atg tat gtc 1563 Ala Thr Ile Met Ser Asp Gly Pro GlyGly Asn Lys Trp Met Tyr Val 430 435 440 ggg aaa cat aaa gct ggc caa gtatgg aga gat atc acc gga aat agg 1611 Gly Lys His Lys Ala Gly Gln Val TrpArg Asp Ile Thr Gly Asn Arg 445 450 455 tct ggt acc gtc acc att aat gcagat ggt tgg ggg aat ttc act gta 1659 Ser Gly Thr Val Thr Ile Asn Ala AspGly Trp Gly Asn Phe Thr Val 460 465 470 aac gga ggg gca gtt tcg gtt tgggtg aag caa taaataagga acaagaggcg 1712 Asn Gly Gly Ala Val Ser Val TrpVal Lys Gln 475 480 485 aaaattactt tcctacatgc agagctttcc gatcactcatacacccaata taaattggaa 1772 gctt 1776 8 516 PRT Bacillus sp. 8 Met LysLeu His Asn Arg Ile Ile Ser Val Leu Leu Thr Leu Leu Leu -30 -25 -20 AlaVal Ala Val Leu Phe Pro Tyr Met Thr Glu Pro Ala Gln Ala His -15 -10 -5-1 1 His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu 510 15 Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala Asn 2025 30 Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp Lys 3540 45 Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp 5055 60 65 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr70 75 80 Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly Ile85 90 95 Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Gly100 105 110 Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg AsnGln 115 120 125 Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys PheAsp Phe 130 135 140 145 Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys TrpArg Trp Tyr His 150 155 160 Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg GlnLeu Gln Asn Lys Ile 165 170 175 Tyr Lys Phe Arg Gly Thr Gly Lys Ala TrpAsp Trp Glu Val Asp Ile 180 185 190 Glu Asn Gly Asn Tyr Asp Tyr Leu MetTyr Ala Asp Ile Asp Met Asp 195 200 205 His Pro Glu Val Ile Asn Glu LeuArg Asn Trp Gly Val Trp Tyr Thr 210 215 220 225 Asn Thr Leu Asn Leu AspGly Phe Arg Ile Asp Ala Val Lys His Ile 230 235 240 Lys Tyr Ser Tyr ThrArg Asp Trp Leu Thr His Val Arg Asn Thr Thr 245 250 255 Gly Lys Pro MetPhe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Ala 260 265 270 Ala Ile GluAsn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe 275 280 285 Asp ValPro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly 290 295 300 305Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His 310 315320 Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly 325330 335 Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr340 345 350 Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe TyrGly 355 360 365 Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met LysSer Lys 370 375 380 385 Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr AlaTyr Gly Thr Gln 390 395 400 His Asp Tyr Phe Asp His His Asp Ile Ile GlyTrp Thr Arg Glu Gly 405 410 415 Asp Ser Ser His Pro Asn Ser Gly Leu AlaThr Ile Met Ser Asp Gly 420 425 430 Pro Gly Gly Asn Lys Trp Met Tyr ValGly Lys His Lys Ala Gly Gln 435 440 445 Val Trp Arg Asp Ile Thr Gly AsnArg Ser Gly Thr Val Thr Ile Asn 450 455 460 465 Ala Asp Gly Trp Gly AsnPhe Thr Val Asn Gly Gly Ala Val Ser Val 470 475 480 Trp Val Lys Gln 4859 40 DNA Artificial sequence Primer DAX-8N 9 gctgcggccg ctgcagatggmttgaayggw acgatgatgc 40 10 43 DNA Artificial sequence Primer DAX-8C 10ggccgtcgac ttattggttc acrtacacgg atacagatcc tcc 43 11 32 DNA Artificialsequence Primer Amrk752-E84Q 11 ctaaggcaca gcttcaacga gctattgggt cc 3212 35 DNA Artificial sequence Primer Amrk752-N96D 12 ccttaaatctaatgatatcg atgtatacgg agatg 35 13 34 DNA Artificial sequence PrimerAmrk752-A315S 13 ttataatttt taccggtctt cacaacaagg tgga 34 14 32 DNAArtificial sequence Primer Amrk752-A445V 14 gtaggacgtc agaatgtaggacaaacatgg ac 32 15 35 DNA Artificial sequence Primer Amrk752-G464N 15ccgttacaat taataacgat ggatggggcg aattc 35 16 34 DNA Artificial sequencePrimer Amrk230-N121D 16 gcaagctgtt caagtagatc caacgaatcg ttgg 34 17 34DNA Artificial sequence Primer Amrk230-N390H 17 gcttgatgca cgtcaagattacgcatatgg cacg 34

1. A variant of a parent alpha-amylase, comprising an alteration at oneor more positions selected from the group of:2,9,14,15,16,26,27,48,49,51,52,53,54,58,73,88,94,96,103,104,107,108,111,114,128,130,133,138,140,142,144,148,149,156,161,165,166,168,171,173,174,178,179,180,181,183,184,187,188,190,194,197,198,199,200,201,202,203,204,205,207,209,210,211,212,214,221,222,224,228,230,233,234,237,239,241,242,252,253,254,255,260,264,265,267,275,276,277,280,281,286,290,293,301,305,314,315,318,329,333,340,341,356,375,376,377,380,383,384,386,389,399,403,404,405,406,427,441,444,453,454,472,479,480 wherein (a) thealteration(s) are independently (i) an insertion of an amino aciddownstream of the amino acid which occupies the position, (ii) adeletion of the amino acid which occupies the position, or (iii) asubstitution of the amino acid which occupies the position with adifferent amino add, (b) the variant has alpha-amylase activity, and (c)each position corresponds to a position of the amino acid sequence ofthe parent alpha-amylase having the amino acid sequence of the SM-36alpaha-amylases shown in SEQ ID NO: 2:
 2. The variant of claim 1, whichvariant has one or more of the following mutations: G2P,A; M9I,L,F;H14Y; L15M,I,F,T; E16P; H26Y,Q,R,N; D27N,S,T; G48A,V,S,T; N49X; Q51X;A52X; D53E,Q,R; V54X; A58V,L,I,F; V73L,I,F; E84Q; G88X; D94X; N96Q;M103I,L,F; N104D; M/L107G,A,V,T,S,I,L,F; G108A; F111G,A,V,I,L,T;A114D,I,L,M,V,R; T125S; D128T,E; S130T,C; Y133F,H; W138F,Y;G140H,R,K,D,N; D142H,R,K,N; S144P; N148S; A149I; R156H,K,D,N; N161X;W165R; D166E; R168P; E171L,I,F; H173R,K,L; I173L; L174I,F;A178N,Q,R,K,H; N179G,A,T,S; T180N,Q,R,K,H; N181X; N183X; W184R,K;D187N,S,T; E188P,T,I,S; N190F; D194X; L197X; G198X; S199X; N200X;I201L,M,F,Y; D202X; F203L,I,F,M; S204X; H205X; E207Y,R; Q209V,L,I,F,M;E210X; E211Q; L212I,F; D214N,R,K,H; D221N; E222Q,T; D224N,Q; Y228F;L230I,F; I233A,V,L,F; K234N,Q; P237X; W239X; T241L,l,F,M; S242P,R;A252T; D253G,A,V,N; Q254K; D255N,Q,E,P; G260A; K264Q,S,T; D265N,Y;V267L,I,F,M; D275N,T; E276K; M277T,I,L,F; E280N,T,Q,S; M281H,I,L,F;V286X, preferably V286Y,L,I,F; Y290X; Y293H,F; S301G,A,D,K,E,R;R305A,K,Q,E,H,D,N; E314K,Q,R,S,T,H,N; A315K,R,S; 1318L,M,F; T329S;E333Q; A340R,K,N,D,Q,E; D341P,T,S,Q,N; G356Q,E,S,T,A; S375P; A376S;K377L,I,F,M; M380I,L,F; E383P,Q; L384I,F; D386N,Q,R,K,I,L; Q389K,R;Y399A,D,H; W403X; D404N; I405L,F; V406I,L,F,A,D; N427X; H441K,N,D,Q,E;R442Q; Q444E,K,R; A445V; Q448A; H453R,K,Q,N; A454S,T,P; G472R,N479Q,K,R; Q480K,R.
 3. The variant of claims 1 or 2, wherein the varianthas the following mutations: N49I+L/M107A; N49L+L/M107A;G48A+N49I+L/M107A; G48A+N49L+L/M107; E188S,T,P+N190F+I201F+K264S;G48A+N49I+L/M107A+E188S,T,P+N190F+I201F+K264S; N190F+I201F; N190F+K264S;I201F+K264S; G140H+D142H+R156H,Y(+S144P); G140K+D142D+R156H,Y(+S144P);L197M+G198Y+S199A; L15T+E188S+Q209V+A376S+G472R;G48A+N49I+L/M107A+G140H+D142H+R156H,Y+E188P+N190F+I201F+K264S(+S144P);N49T+L/M107A+G140H+D142H+R156H,Y+E188P+N190F+I201F+K264S(+S144P);
 4. Thevariant according to any of claims 1-3, wherein the parent alpha-amylasehas an amino acid sequence which has a degree of identity to SEQ ID NO:2 of at least 60%, preferably 70%, more preferably at least 80%, evenmore preferably at least about 90%, even more preferably at least 95%,even more preferably at least 97%, and even more preferably at least99%.
 5. The variant of any of claims 14, wherein the parentalpha-amylase is encoded by a nucleic acid sequence, which hydridizesunder medium, preferred high stringency conditions, with the nucleicacid sequence of SEQ ID NO: 1 or
 3. 6. The variant of claims 1-5,wherein the parent alpha-amylase is KSM-K38 shown in SEQ ID NO:4.
 7. Thevariant of claims 1-6, which variant has altered pI, in particular ahigher pI than the parent alpha-amylase.
 8. A DNA construct comprising aDNA sequence encoding an alpha-amylase variant according to any one ofclaims 1 to
 7. 9. A recombinant expression vector which carries a DNAconstruct according to claim
 8. 10. A cell which is transformed with aDNA construct according to claim 8 or a vector according to claim
 9. 11.A cell according to claim 10, which is a microorganism, preferably abacterium or a fungus, in particular a gram-posifive bacterium, such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus or Bacillus thuringiensis.
 12. A detergent additive comprising analpha-amylase variant according to any one of claims 1-7, optionally inthe form of a non-dusting granulate, stabilised liquid or protectedenzyme.
 13. A detergent additive according to claim 12, which contains0.02-200 mg of enzyme protein/g of the additive.
 14. A detergentadditive according to claims 12 or 13, which additionally comprisesanother enzyme such as a protease, a lipase, a peroxidase, a pectatelyase, an amylase, or another amylolytic enzyme, such as maltogenicalpha-amylase or glucoamylase, mannanase, CGTase, and/or a cellulase.15. A detergent composition comprising an alpha-amylase variantaccording to any of claims 1-7.
 16. A detergent composition according toclaim 15, which additionally comprises another enzyme such as aprotease, a lipase, a peroxidase, a pectate lyase another amylolytcenzyme, glucoamylase, CGTase, mannanase, maltogenic amylase, and/or acellulase.
 17. A manual or automatic dishwashing detergent compositioncomprising an alpha-amylase variant according to any of claims 1-7. 18.A dishwashing detergent composition according to claim 17, whichadditionally comprises another enzyme such as a protease, a lipase, aperoxidase, a pectate lyase, an amylase, or another amylolytic enzyme,such as glucoamylase, CGTase, mannanase, maltogenic amylase and/or acellulase.
 19. A manual or automatic laundry washing compositioncomprising an alpha-amylase variant according to any of claims 1-7. 20.A laundry washing composition according to claim 19, which additionallycomprises another enzyme such as a protease, a lipase, a peroxidase, apectate lyase, an amylase, and/or another amylolytic enzyme, such asglucoamylase, CGTase, mannanase, maltogenic amylase and/or a cellulase.21. Use of an alpha-amylase variant according to any one of claims 1-7or a composition or an additive according to claims 12 to 20 for washingand/or dishwashing.
 22. Use of an alpha-amylase variant according to anyone of claims 1-7 or a composition or an additive according to claims 12to 20 for textile desizing.
 23. Use of an alpha-amylase variantaccording to any of claims 1-7 or a composition or an additive accordingto claims 12 to 20 for starch liquefaction.
 24. Use,of an alpha-amylasevariant according to any of claims 1-7 or a composition or an additiveaccording to claims 12 to 20 for alcochol production, in particularethanol production.